C. Altenbach et al., STRUCTURAL FEATURES AND LIGHT-DEPENDENT CHANGES IN THE CYTOPLASMIC INTERHELICAL E-F LOOP REGION OF RHODOPSIN - A SITE-DIRECTED SPIN-LABELING STUDY, Biochemistry, 35(38), 1996, pp. 12470-12478
Thirty consecutive single cysteine substitution mutants in the amino a
cids Q225-I256 of bovine rhodopsin have been prepared and modified wit
h a sulfhydryl specific nitroxide reagent. This sequence includes the
E-F interhelical loop, a transducin interaction site. The accessibilit
ies of the attached nitroxides to collisions with hydrophilic and hydr
ophobic paramagnetic probes in solution were determined, and the elect
ron paramagnetic resonance spectra analyzed in terms of side chain mob
ility, both in the dark and after photoactivation. Accessibility data
shows that the rhodopsin polypeptide chain crosses an aqueous/hydropho
bic boundary in the range V227-K231 and again in the range V250-V254.
In the hydrophobic segments, both the accessibility and mobility data
are consistent with helical structures. In the regions of the sequence
located within the aqueous phase, periodic variation in both accessib
ility and mobility of the spin-labeled side chains indicates that the
E-F interhelical loop is largely alpha-helical, being formed by regula
r extensions of the E and F helices by about 1.5 and 3 turns, respecti
vely. Judging from nitroxide mobilities, the putative extension of hel
ix E in the aqueous phase is more dynamic than that of helix F. Change
s in the electron paramagnetic resonance characteristics of the spin-l
abeled rhodopsin upon photoactivation indicate that chromophore isomer
ization results in patterns of structural changes that can be interpre
ted in terms of movements of helices that extend into the aqueous loop
regions.